Cyanine dyes are widely used in various fields, and they are also used in the field of fluorescence imaging for studying physiological functions as fluorescence labels of biological molecules. In particular, tricarbocyanine type dyes have a maximum absorption wavelength and maximum fluorescence wavelength in the near infrared region of around 650 to 950 nm, of which lights are relatively less absorbed by biological molecules, and thus they have an advantage that they allow use of lights of a wavelength which can penetrate into deep parts of biological tissues. In addition, biological substances scarcely emit autofluorescence of the near infrared region. More specifically, the characteristics of tricarbocyanine type dyes are suitable for in vivo imaging. In addition to cyanine type dyes for directly labeling biological molecules with fluorescence, tricarbocyanine dyes which specifically react with biological molecules to change fluorescence intensity thereof have recently been developed. One of them is a near infrared fluorescent probe for calcium ions (Ozmen, B., et al., Tetrahedron Lett., 41, pp. 9185-9188, 2000), and another is a near infrared fluorescent probe for nitrogen monoxide (NO) (WO2005/080331). These fluorescent probes give only fluorescence intensity changes, but do not show changes of excitation wavelength and fluorescence wavelength, before and after a specific reaction with a biological molecule.
It is known that intracellular pH is maintained at about 6.8 to 7.4 in cytoplasm, or maintained to be acidic, i.e., about 4.5 to 6.0, in lumens of organelles such as Golgi apparatuses, small granule vesicles, coated vesicles, endosomes, and lysosomes, and that such pH changes in association with various cell responses. Such change of intracellular pH controls various cell functions, and there are various reports especially relating to physiological roles thereof in apoptosis, endocytosis, homeostasis, ion transport, and the like (Rich, I. N., et al., J. Cell Physiol., 177 (1) 109, 1998; Meisenholder G. W., et al., J. Biol. Chem., 271, 16260, 1996). Therefore, measurement of intracellular pH is important for understanding control mechanisms of intracellular reactions.
In order to measure intracellular pH, compounds which are protonated or deprotonated depending on intracellular pH, and emit fluorescence in response to the change (pH fluorescent probe) have conventionally been used. As pH fluorescent probes having the fluorescein structure as a mother nucleus, for example, BCECF (2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein) and derivatives thereof, CFDA (carboxyfluorescein diacetate) and derivatives thereof, SNARF-1 (seminaphthorhodafluor) and derivatives thereof (“Handbook of Fluorescent Probes and Research Chemicals”, 10th Edition by Richard P. Haugland, chapter 20, “pH indicators”, which is a catalogue of Molecular Probe, for all the compounds) and the like have been put into practical use. As pH probes having the cyanine structure as a mother nucleus, the compounds described in International Patent Publication WO00/75237 and CypHer (GE Healthcare Bioscience) are available.
However, BCECF and CFDA, which are pH fluorescent probes having the fluorescein structure, have an excitation wavelength not longer than 550 nm, which results in low permeability for biological tissues, and therefore they have problems in that they cannot achieve observation of deep parts of tissues, and they are readily influenced by autofluorescence of biological substances (fluorescence emitted by NADH, flavins and the like) at measurement. Further, CypHer and the pH fluorescent probes described in International Patent Publication WO00/75237 have the cyanine structure, and they emit fluorescence when the nitrogen atom of the nitrogen-containing hetero aromatic ring bonded to the polymethine chain of the cyanine structure is protonated. These fluorescent probes enable measurement with an excitation light of a long wavelength not shorter than 640 nm, and fluorescence intensity thereof increases along with the decrease of pH in the neutral region or lower pH region. Therefore, they have an advantage that they are hardly influenced by autofluorescence of biological substances. However, application of fluorescent probes to cells involves many factors which affect the measurement. For example, concentration of a fluorescent probe introduced into cells may vary depending on type of the cells, fluorescence intensity may vary depending on thickness of cell membrane even in a measurement region, a fluorescent probe may localize at a highly hydrophobic portion such as membranes, and the like.
As a method for reducing measurement errors induced by these factors to realize accurate quantitative analysis, the ratio method has been developed and used (Kawanishi Y., et al, Angew. Chem. Int. Ed., 39(19), 3438, 2000). This method comprises the step of measuring fluorescence intensities at two different wavelengths in a fluorescence spectrum or an excitation spectrum to detect a ratio thereof. In this method, influence of concentration of the fluorescent probe itself or intensity of excitation light can be neglected, and measurement errors can be eliminated, which may be caused by localization of the fluorescent probe itself, change of concentration thereof, discoloration thereof, or the like, when the measurement is performed at one wavelength.
For example, SNARF-1, which is a pH fluorescent probe having the fluorescein structure, has a property that the peak of fluorescence wavelength shifts to the longer wavelength side due to deprotonation when pH shifts to the alkaline side, and when the compound is excited around 500 nm, fluorescence intensity around 580 nm decreases along with the increase of pH, whilst fluorescence intensity around 640 nm increases with the increase of pH. Therefore, if this compound is excited at an appropriate wavelength around 500 nm, and ratio of fluorescence intensities at appropriate two wavelengths around 580 to 640 nm, pH can be accurately measured regardless of probe concentration, intensity of light source, size of cells, and the like. However, there has not been known any fluorescent probe which enables imaging of intracellular pH change by the ratio method using an excitation light of the near infrared region around 650 to 950 nm, which has superior permeability for biological tissues, and thus it has been desired to develop a fluorescent probe for measuring fluorescence of the near infrared region around 650 to 950 nm by the ratio method, which fluorescence is less influenced by autofluorescence of biological substances and has superior permeability for biological tissues, in order to accurately perform fluorescence imaging of pH change.